The present invention relates to a culture media for culturing neurons, to methods for preparing the culture media, and to methods for culturing neurons by use of the culture media.
In order to culture neurons ex vivo, many studies have been conducted since long ago. The discovery of the nerve growth factor (NGF), being the first discovery of a factor that specifically functions on neurons, was epoch-making in the field of neurobiology. In culturing neurons, NGF has made it possible to induce growth of nerve fibers, and accordingly, neurons have come to be cultured under conditions more closely resembling in vivo.
Recently, new nerve growth factors such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-5 (NT-5), and glial cell line derived neurotrophic factor (GDNF) have been discovered one after another. These factors have been studied with respect to their effects and functional mechanisms by use of cultured neurons, and have now come to the stage where use thereof as medicines for the treatment of diseases is being considered through the application of genetic engineering techniques that have been put into practice.
However, when these factors are investigated in an ordinary culture system for central nerve cells, a disadvantage is encountered in that a plurality of substances must be added in order for a significant effect to be obtained for various neurons; i.e., when a single substance is added, effect is observed on only certain specific cells.
Regarding culturing of central nerve cells, there has been an another approach in which use of hormones (insulin, thyroxine, progesterone, etc.), vitamins, unsaturated fatty acids, and growth factors (such as basic fibroblast growth factor) has been studied. Serum-free culture media in which these substances are combined differently have heretofore been reported [Journal of Neuroscience Methods, 23:75 (1988), etc.]. Although these culture media are effective toward certain cell lines (such as the glial cell line), there are cases in which neurons cannot be stably cultured, or in which the culture media are effective toward neurons but proliferation of glial cells is simultaneously stimulated, to thereby render the culture system a so-called mixed culture system involving glial cells in addition to neurons. This is detrimental, particularly when pharmacological action on neurons is to be studied. For example, although the N2 supplement of Bottenstein et al. [insulin (5 xcexcg/ml), transferrin (100 xcexcg/ml), progesterone (20 nM), putrescine (100 xcexcM), and selenious acid salts (30 nM); Proceeding of National Academy of Science, U.S.A., 76:514(1979)] is suitable for culturing glial cell lines, it cannot stably maintain viable primary neurons. Also, the culture medium disclosed by Brewer et al. [Brain Research, 65:494(1989)] cannot stably maintain cellular functions when used for long-term culturing, although it permits short-term culturing of central nerve cells.
In the meantime, it is known that a culture supernatant of a glial cell line or primary glial cells is used for culturing neurons. When a culture supernatant of a glial cell line is used, it is generally unavoidable that the supernatant acts not only on neurons but also on glial cells to thereby stimulate the proliferation of glial cells. In other words, although the culture supernatant is effective in maintaining survival of neurons, it exhibits much greater effects on glial cells. A so-called glial cell growth factor has been purified from these culture supernatants [Journal of Biological Chemistry, 268: 2857 (1993), etc.].
In addition, Japanese Patent Application Laid-Open (kokai) No. 7-101990 discloses that a concentrated culture supernatant of astrocytoma, which is a type of glial cell lines, exhibits 30-50% elevation in activity of cultured neurons. In that publication, it is also disclosed that the concentrate suppresses proliferation of neuroblastoma. The technique of that publication is significant in that a culture supernatant is concentrated to take up part of active components so as to suppress proliferation of glial cells, which proliferation is the drawback involved in use of a culture supernatant of a glial cell line. However, in order to stably and consistently culture neurons, collective action of a plurality of components is required rather than the action of a single substance. A stable culture system for neurons cannot be established through elevation in activity as low as 30% relative to the case in which the concentrate is not used.
In a method in which primary glial cells are used, an ordinary serum-free culture medium is employed, or serum-containing culture medium is used to obtain a culture supernatant. When an ordinary serum-free culture medium is used to obtain a culture supernatant, the culture medium does not satisfactorily promise viability of glial cells, and therefore, stable culturing cannot be realized. Consequently, the amount of factors (substances) that act on the neurons present in the culture supernatant becomes small, to thereby exhibit only an insignificant effect in culturing of neurons. Moreover, collection of a culture supernatant may be performed in limited numbers, and after several times of collection, it becomes difficult to collect a culture supernatant that provides a stable effect. On the other hand, when a serum-containing culture medium is used, a culture supernatant can be collected in a stable manner. However, when neurons are cultured, the growth factor acts on glial cells rather than on co-existing neurons, to thereby stimulate proliferation of cells. As a result, stable culturing of neurons is hampered.
Generally, serum-free culture media are supplemented with trophic factors such as hormones. For example, Japanese Patent Application Laid-Open (kokai) No. 3-66700 discloses a culturing method in which Dulbeccol""s modified Eagle medium (hereinafter referred to as DMEM) is supplemented with insulin (5 xcexcg/ml), transferrin (1 xcexcg/ml), hydrocortisone (20 nM), and 3,3xe2x80x2,5-triiodo-L-thyronine (0.3 nM). However, when this culture supernatant is used for the culturing of neurons, stable culturing cannot be achieved beyond a period of several days. Also, xcex12-macroglobulinxe2x80x94which is explicitly described in that publication as a neurite-outgrowth promoting factorxe2x80x94exhibits insignificant effect on achieving stable culturing for central nerve cells, and thus plays only an auxiliary role, not a principal role.
Japanese Patent Application Laid-Open (kokai) No. 3-155777 discloses that a certain factor produced by microgliacytes is effective for neurite-outgrowth. Microglia is said to exhibit a function analogous to lymphocyte such as macrophage, and their activity elevates when tissue is injured. However, it is accepted that macrogliasxe2x80x94including astroglial cellsxe2x80x94are predominantly present in living bodies in general, and that it is principally macroglias that maintain homeostasis except in special cases such as inflammation.
The above-described conventional culture media are not sufficiently effective when they are used to culture central nerve cells, and therefore, consistent culturing of neurons cannot be achieved. In other words, satisfactory results as expected cannot be obtained through use of conventional culture media in neuropharmacological tests. Accordingly, the present invention was made in view of the foregoing circumstances in relation to culturing of neurons, and objects of the invention are to provide culture media that are capable of consistently culturing neurons for long periods.
Based on the idea that the presence of a variety of trophic factors must be required for consistent culturing of neurons, the present inventors investigated how trophic factors affect culturing of neurons. They also examined a culture supernatant of primary astroglial cells as one of the trophic factors. As a result, the trophic factors they examined were found to provide insufficient effects when used independently and solely. Particularly, it was found that the culture supernatant of primary astroglial cells was in fact not as effective as had been reported toward consistent culture of neurons. Therefore, the present inventors initiated investigations of combination effects and synergetic effects through use of a plurality of trophic factors, and conducted careful studies on different effects of culturing, thus leading to completion of the present invention.
Accordingly, the present invention provides culture media for neurons characterized by comprising (A) a culture supernatant obtained from a culture of primary astroglial cells in a trophic medium supplemented with insulin and transferrin, and (B) albumin.
The present invention also provides a method for preparing the above-described culture medium for neurons characterized by culture and proliferation of primary astroglial cells in a medium supplemented with animal serum, and culturing in a trophic medium supplemented with insulin and transferrin, then collection of a supernatant of the culture, and addition of albumin to the obtained culture supernatant.
The present invention further provides a method for culturing neurons characterized by culturing neurons in the above-described culture media for neurons.
In order to prepare a culture medium for neurons, astroglial cells are collected from the animal brain. The collected astroglial cellsxe2x80x94primary astroglial cellsxe2x80x94are proliferated until they come to be in sufficient amounts. The astroglial cells are preferably collected from the cerebrums of newborn animals (1-2 days old). Animals suitable for the collection of astroglial cells include rats, mice, bovines, horses, pigs, monkeys, rabbits, and hens, with rats and mice being particularly preferred.
Briefly, the cerebrum is excised out of the brain of a newborn, and after the cerebral meninges is removed therefrom, the cerebrum cells are dissociated through use of enzymes such as trypsin, disperse, collagenase, and papain. Use of 0.05-0.35 w/v % (hereinafter simply referred to as %) trypsin is particularly preferred. It is also effective to add, to trypsin, 0.01% (100-500 U/ml) deoxyribonuclease or 0.01% ethylenediamine-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EDTA).
The collected astroglial cells are preferably cultured and proliferated in a medium containing animal serum. The animal serum is preferably bovine serum, and more preferably, fetal calf serum, calf serum, or neonatal calf serum. The amount of animal serum to be added is preferably in the range of 5-20%. The medium is not particularly limited so long as it is a trophic medium for culturing animal cells. Examples of such medium include Eagle""s minimum essential medium (hereinafter abbreviated as MEM), Dulbecco""s modified Eagle medium (hereinafter abbreviated as DMEM), DMEM/HAM""s F-12 medium (hereinafter abbreviated as F-12), F-12, and HAM""s F-10 medium (hereinafter abbreviated as F-10). Although the mixing ratio of DMEM to F-12 in DMEM/F-12 media may vary, the ratio be preferably in the range from 60/40 to 40/60 (on a weight basis) so that the resultant media have the traits of both media.
The astroglial cells dispersed in a culture medium are cultured until confluency through use of a flask, a dish, or a platexe2x80x94all of which are used for cell culturexe2x80x94or a polylysine-coated flask, a polylysine-coated dish, a polylysine-coated plate, or a polylysine-coated microcarrier. The culture area is preferably 10-100 cm2 with respect to one newborn animal. Subsequently, subculturing is performed until confluency. In subculturing, a culture area of 2-10 times is preferred. The cells that reached confluency are mainly those categorized as type 1 astroglial cell (or type 1 astrocyte). That the cells are type 1 astroglial cell is confirmed by immunocytochemical staining. The cells are stained with anti-GFAP (GFAP: glial fibrillary acidic protein) antibody, but are not stained with anti-A2B5 antibody (anti-sialosyl glycoprotein antibody).
When the cells reach confluency, the culture medium is discarded and the residue is washed with phosphate buffered saline, etc.
The thus-obtained primary astroglial cells are cultured in a trophic medium supplemented with insulin and transferrin, and their supernatant is collected. Trophic media which may be used herein may be one or more members selected from the group consisting of MEM, DMEM, F-10, and F-12. Preferred media are MEM, DMEM, DMEM/F-10 and DMEM/F-12, with DMEM/F-12 being particularly preferred. The preferred ratio of DMEM to F-12 is 60/40-40/60 (on a weight basis).
These trophic media are supplemented with insulin and transferrin. Preferably, selenious acid or a salt thereof is additionally incorporated. Insulin is added in an amount so as to achieve an insulin concentration of 1-100 xcexcg/ml, and preferably 3-20 xcexcg/ml. Transferrin is added in an amount so as to achieve a transferrin concentration of 1-100 xcexcg/ml, and preferably 3-20 xcexcg/ml. Examples of salts of selenious acid include sodium selenite and potassium selenite. It is preferred that selenious acid or a salt thereof be added in such an amount that will make its concentration 1-100 nM; particularly preferably 3-50 nM. All of insulin, transferrin, and salts of selenious acid are water-soluble, and therefore, they may be incorporated as they are. It is also an advantageous approach that a solution containing any of these components at a high. concentration is prepared in advance and a certain amount thereof is incorporated.
The culture period is as short as one day. If a new medium and supplements are replenished after supernatant has been collected to thereby perform culture, supernatant can be collected repeatedly. In this way, supernatant can be collected every day for more than 10 times (days) up to 15 times (days) for a single preparatory operation of primary astroglial cells.
The culture supernatant thus collected is preferably used after being sterilized by being passed through a filter having pores of 0.02-0.45 xcexcm to thereby remove cell debris.
When the culture supernatant alone is used as a culture medium, neurons cannot be cultured stably. Stable culture is possible only after albumin is added to the culture medium. Preferably, progesterone is also added in addition to albumin. The amount of albumin to be incorporated is preferably such that will make the albumin concentration 0.5-2.5 mg/ml. The amount of progesterone is preferably such that will make the progesterone concentration 1-100 nM.
When it is found that the concentration of insulin, transferrin, or selenious acid or a salt thereof is excessively low, it is preferred that these components be added so as to adjust the concentration of insulin to 1-100 xcexcg/ml, that of transferrin to 1-100 xcexc/ml, and that of selenious acid or a salt thereof to 1-100 nM.
Preferably, the culture supernatant additionally contains a combination of superoxide dismutase and catalase and/or xcex1-tocopherols. Preferred concentrations of these supplements are 1-100 xcexcg/ml for superoxide dismutase, 1-100 xcexcg/ml for catalase, and 1-100 xcexcg/ml for xcex1-tocopherols. Examples of xcex1-tocopherols include xcex1-tocopherol and esters of xcex1-tocopherol such as tocopherol acetate and tocopherol succinate.
When culture media supplemented with albumin and other supplements as described above are used, neurons can be cultured in a consistent manner. Also, the culture media of the invention successfully overcome the problem of unstable culture which cannot be avoided when low-density culture is performed with previous culture media.
To the culture media may be newly added any of the aforementioned media as a trophic source for neurons. An example of such media is a DMEM/F-12 medium mixture. The medium is preferably added so as to achieve an amount of 0-75%, and particularly preferably 0.1-50%.
Examples of particularly preferred combinations of supplements include the following: albumin, progesterone, insulin, transferrin, and selenious acid (or a salt thereof); these five species plus xcex1-tocopherols; the five species plus superoxide dismutase and catalase; the five species plus xcex1-tocopherols, superoxide dismutase and catalase; and any of these combinations plus a DMEM/F-12 medium mixture.
In order to incorporate the above-described supplements into a culture supernatant, a method similar to that described above may be used if water-soluble progesterone and water-soluble xcex1-tocopherol are used for progesterone and xcex1-tocopherol, respectively. That is, a highly concentrated solution containing the additives may be prepared in advance and added. If water-insoluble progesterone and water-insoluble xcex1-tocopherol are used, they are dissolved in ethanol in advance.
A culture supernatant can be stored stably in a frozen state. Therefore, in the case in which supernatants are collected every day consecutively, the supernatant collected each time is frozen without being combined with supplements such as insulin; and after a plurality of supernatants have been collected and frozen, they are thawed, uniformly mixed, and then combined with the supplements. This procedure provides more uniform culture media for neurons. It is preferred that the supplements in this case be prepared into a solution state before being added.
The supplements are stably stored if dissolved in pure water or in an aqueous solution such as phosphate buffered saline and then frozen. Progesterone, xcex1-tocopherol, and other water-insoluble components are stored in a frozen state after they are dissolved in ethanol, etc. Also, the culture media of the present invention prepared as described above can be stably stored in a frozen state. The temperature at which any of culture supernatant, supplements, and blended culture media are stored in a frozen state is preferably between xe2x88x9210 and xe2x88x9280xc2x0 C. If the storage temperature is that of a typical refrigerator (4-8xc2x0 C.), it is difficult to store them stably for long periods. Preferably, a cycle of freezing and thawing should not be repeated.
In order to culture neurons through use of the culture media of the present invention, neurons are added to the culture media and a conventional culture procedure is performed. Neurons can be prepared by use of the animals similar to those from which primary astroglial cells are prepared. Although culture is possible with neonatal individuals, viability of neurons is generally improved when fetal animals are used. If rats are used, embryos of 15-20 days old are preferred. It is also possible to use more immature embryos. Moreover, neurons in limited regions of the brain, such as hippocampus, corpus striatum, septum, midbrain, or cerebellum may be cultured. When neurons in the cerebellum are desired to be cultured, good results can be obtained through use of neonatal individuals of approximately 1 week old in accordance with the differentiation of neurons.
In the case in which the brain is treated with an enzyme so as to dissociate neurons, glial cells (such as primary astroglial cells) are generally included in addition to neurons. Astroglial cells are categorized as type 1 and type 2, and when serum-added culture media are concerned, type 1 astroglial cells are grown to cause a problem. The culture media of the present invention are effective in suppressing the proliferation of these cells. Although types 1 and 2 astroglial cells cannot be definitely distinguished from each other based on the shape of primary astroglial cells,they are distinguished through use of immunocytochemical staining. Both types 1 and 2 are stained with anti-GFAP antibody, but only type 2 is stained with anti-A2B5 antibody. This difference in staining property can distinguish these two types of astroglial cells.
When the fetus brain is used, the proportion of undifferentiated cells is significant. The culture medium of the present invention induces proliferation and differentiation of oligodendroglia (or oligodendrocyte)xe2x80x94type 2 astroglia (or type 2 astrocyte) stem cells (hereinafter referred to as O-2A stem cells), which are one type of the undifferentiated stem cells. When incubation is continued, oligodendrogliasxe2x80x94which are initially not observedxe2x80x94will come to be observed in the culture system. They are O-2A stem cells that have been proliferated and differentiated. The fact that the cells are oligodendroglias can be confirmed by use of similar immunocytochemical staining that is used for distinguishing astroglial cells. For example, when anti-GC (galactocerebroside) antibody, anti-MBP (myelin basic protein) antibody, etc. are used for staining, the oligodendroglia can be clearly distinguished.
A specific example of a culture method for neurons is as follows. The brain is removed in a manner similar to that as described above. A neuron dissociation is prepared through use of an enzyme such as trypsin, papain, or disperse. Preferably, papain (10-50 U/ml) is used. Lcystein (0.5-5 mM) and glucose (5-50 mM) are added to phosphate buffered saline in which papain has been dissolved. The brain tissue is enzymatically treated with the resultant solution at 37xc2x0 C. for 30-120 minutes. The enzymatic solution is carefully stirred and mixed to thereby disperse the neurons. If deoxyribonuclease (0.01%) is further added, there can be prevented agglutination of cells due to the presence of nucleic acid that has been leached.
Subsequently, cells are separated by use of a centrifugal separator. The separated cells are added to the culture media of the present invention prepared as described above so as to prepare a cell suspension of 10,000-2,000,000 cells/ml. The cell suspension is placed in a culture plate or a culture dish and is cultured in a 5% CO2 gas incubator at 37xc2x0 C. The material of the plate, dish, etc. is not particularly limited, and may be glass, plastic, etc. Preferably, the plate or dish is coated with a single layer or a plurality of layers of polylysine, polyornithine, polyallylamine, protamine, laminin, collagen, gelatin, fibronectin, vitronectin, tenascin, or a mixture of them.